Unprocessed Human Plasma Research Articles

Dual-functionalized architecture enables stable and tumor cell-specific SiO2NPs in complex biological fluids.

Most commercial anticancer nanomedicines are administered intravenously. This route is fast and precise as the drug enters directly into the systemic circulation, without undergoing absorption processes. When nanoparticles come into direct contact with the blood, however, they interact with physiological components that can induce colloidal destabilization and/or changes in their original biochemical identity, compromising their ability to selectively accumulate at target sites. In this way, these systems usually lack active targeting, offering limited therapeutic effectiveness. In the literature, there is a paucity of in-depth studies in complex environments to evaluate nanoparticle stability, protein corona formation, hemolytic activity, and targeting capabilities. To address this issue, fluorescent silica nanoparticles (SiO2NPs) are here functionalized with zwitterionic (kinetic stabilizer) and folate groups (targeting agent) to provide selective interaction with tumor cell lines in biological media. The stability of these dually functionalized SiO2NPs is preserved in unprocessed human plasma while yielding a decrease in the number of adsorbed proteins. Experiments in murine blood further proved that these nanoparticles are not hemolytic. Remarkably, the functionalized SiO2NPs are more internalized by tumor cells than their healthy counterparts. Investigations of this nature play a crucial role in garnering results with greater reliability, allowing the development of nanoparticle-based pharmaceutical drugs that exhibit heightened efficacy and reduced toxicity for medical purposes.

Surface Oxygen Deficiency Enabled Spontaneous Antiprotein Fouling in WO3 Nanosheets for Biosensing in Biological Fluids.

Biofouling deteriorates the performance of sensors operated in biofluids. Protein adsorption is believed to be the first step of biofouling, which also reduces biocompatibility by further inducing cell adhesion, platelet activation, and even inflammation. Current studies of antifouling coatings are focused on polymers and hydrogels, which have succeeded in remaining resistant to protein adsorption, but their application on sensor electrodes is limited due to low conductivity and biocompatibility. Here, we report a spontaneous antibiofouling strategy for sensor electrodes by controlling oxygen vacancies in WO3 nanosheets. Irreversible adsorption of proteins was reduced by 76% in unprocessed human plasma when electrodes were coated with WO3 rich in surface oxygen vacancy. These electrodes maintained 91% of the initial current density after 1 month of incubation in human plasma.

Electrochemical sensing of doxorubicin hydrochloride under sodium alginate antifouling conditions using silver nanoparticles modified glassy carbon electrodes

Doxorubicin (DOX) is a highly effective anticancer drug with a narrow therapeutic window; thus, sensitive and timely detection of DOX is crucial. Using electrodeposition of silver nanoparticles (AgNPs) and electropolymerization of alginate (Alg) layers on the surface of a glassy carbon electrode, a novel electrochemical probe was constructed (GCE). The fabricated AgNPs/poly-Alg-modified GCE probe was utilized for the quantification of DOX in unprocessed human plasma samples. For the electrodeposition of AgNPs and electropolymerization of alginate (Alg) layers on the surface of GCE, cyclic voltammetry (CV) was used in the potential ranges of −2.0 to 2.0 V and −0.6 to 0.2 V, respectively. The electrochemical activity of DOX exhibited two oxidation processes at the optimum pH value of 5.5 on the surface of the modified GCE. The DPV spectra of poly(Alg)/AgNPs modified GCE probe toward consecutive concentrations of DOX in plasma samples demonstrated wide dynamic ranges of 15 ng/mL-0.1 μg/mL and 0.1–5.0 μg/mL, with a low limit of quantification (LLOQ) of 15 ng/mL. The validation results indicated that the fabricated electrochemical probe might serve as a highly sensitive and selective assay for the quantification of DOX in patient samples. As an outstanding feature, the developed probe could detect DOX in unprocessed plasma samples and cell lysates without the requirement for pretreatment.

An ultrasensitive and preprocessing-free electrochemical platform for the detection of doxorubicin based on tryptophan/polyethylene glycol-cobalt ferrite nanoparticles modified electrodes

Doxorubicin (DOX) is an anticancer drug which can effectively inhibit the growth of cancer cells and aids the immune-mediated elimination of tumoral cells. Developing a new technique for analysis of DOX in clinical fluids is highly required. Here, a novel electrochemical sensor was designed using tryptophan (Trp)/(polyethylene glycol)PEGylated-CoFe2O4 nanoparticles to modify glassy carbon electrodes’ surface and was utilized to determine DOX in unprocessed human plasma samples. PEGylated-CoFe2O4 nanoparticles were coated on the surface of the glassy carbon electrode to provide the PEGylated-CoFe2O4/GCE probe. Under optimized conditions, the low limit of quantification (LLOQ) of the proposed sensor was 30 ng/mL and the linear ranges for the determination of DOX were 30 ng/mL to 1.0 μg/mL and 1.0 μg/mL to 5.0 μg/mL, respectively. PEG molecules provided an antifouling effect to prevent precipitation of the macromolecules on the surface of the fabricated electrode. Obtained results indicated that the suggested electrochemical sensor can be utilized for specific and sensitive determination of DOX in plasma samples.

An imaging flow cytometry-based methodology for the analysis of single extracellular vesicles in unprocessed human plasma

Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. To rule out selection bias or introduction of artefacts caused by EV isolation techniques, we present a clinically feasible, imaging flow cytometry (IFCM)–based methodology to phenotype and determine the concentration of EVs with a diameter ≤400 nm in human platelet-poor plasma (PPP) without prior isolation of EVs. Instrument calibration (both size and fluorescence) were performed with commercial polystyrene beads. Detergent treatment of EVs was performed to discriminate true vesicular events from artefacts. Using a combination of markers (CFSE & Tetraspanins, or CD9 & CD31) we found that >90% of double-positive fluorescent events represented single EVs. Through this work, we provide a framework that will allow the application of IFCM for EV analysis in peripheral blood plasma in a plethora of experimental and potentially diagnostic settings. Additionally, this direct approach for EV analysis will enable researchers to explore corners of EVs as cellular messengers in healthy and pathological conditions.

Ultrarapid Method for Coating Electrochemical Sensors with Antifouling Conductive Nanomaterials Enables Highly Sensitive Multiplexed Detection in Whole Blood.

The commercialization of electrochemical (EC)-sensors for medical diagnostics is currently limited by their rapid fouling in biological fluids, and use of potential antifouling coatings is hindered by the complexity and cost of application methods. Here, a simple ultrafast (< 1min) method is described for coating EC-sensors with cross-linked bovine serum albumin infused with conductive, pentaamine-functionalized, graphene particles that can be stored at room temperature for at least 20-weeks, which provides unprecedented sensitivity and selectivity for diagnostic applications. The antifouling coating is applied directly on-chip using rapid heating via simple dip-coating, which provides unprecedented high levels of electrode conductivity for up to 9-weeks in unprocessed biological samples. This method is leveraged to develop a multiplexed platform for detecting clinically relevant biomarkers including myocardial infarction and traumatic brain injury using only 15µL of blood. Single-digit pg mL-1 sensitivity is obtained within minutes in unprocessed human plasma and whole blood, which is faster and at least 50 times more sensitive than traditional enzyme-linked immunosorbent assays, and the signal generated is stable enough to be measured after 1 week of storage. The multiplexed EC-sensor platform is validated by analyzing 22 patient samples and demonstrating excellent correlation with reported clinical values.

In Situ Nanocoating on Porous Pyrolyzed Paper Enables Antibiofouling and Sensitive Electrochemical Analyses in Biological Fluids.

Electrochemical detection in complex biofluids is a long-standing challenge as electrode biofouling hampers its sensing performance and commercial translation. To overcome this drawback, pyrolyzed paper as porous electrode coupled with the drop casting of an off-the-shelf polysorbate, that is, Tween 20 (T20), is described here by taking advantage of the in situ formation of a hydrophilic nanocoating (2 nm layer of T20). The latter prevents biofouling while providing the capillarity of samples through paper pores, leveraging redox reactions across both only partially fouled and fresh electrodic surfaces with increasing detection areas. The nanometric thickness of this blocking layer is also essential by not significantly impairing the electron-transfer kinetics. These phenomena behave synergistically to enhance the sensibility that further increases over long-term exposures (4 h) in biological fluids. While the state-of-the-art antibiofouling strategies compromise the sensibility, this approach leads to peak currents that are up to 12.5-fold higher than the original currents after 1 h exposure to unprocessed human plasma. Label-free impedimetric immunoassays through modular bioconjugation by directly anchoring spike protein on gold nanoparticles are also allowed, as demonstrated for the COVID-19 screening of patient sera. The scalability and simplicity of the platform combined with its unique ability to operate in biofluids with enhanced sensibility provide the generation of promising biosensing technologies toward real-world applications in point-of-care diagnostics, mass testing, and in-home monitoring of chronic diseases.

Chemical binding of horseradish peroxidase enzyme with poly beta-cyclodextrin and its application as molecularly imprinted polymer for the monitoring of H2 O2 in human plasma samples.

In this study, a selective and sensitive molecular imprinting-based electrochemical sensors, for horseradish peroxidase (HRP) entrapment was fabricated using electro polymerization of ß-Cyclodextrin (ß-CD) on the surface of glassy carbon electrode. Poly beta-cyclodextrin P(ß-CD) provide efficient surface area for self-immobilization of HRP as well as improve imprinting efficiency. The proposed imprinted biosensor successfully utilized for detection of HRP with excellent analytical results which linear range is 0.1 mg/mL to 10 ng/mL with LOD of 2.23 ng/mL. Furthermore, electrocatalytical activity of the prepared biosensor toward the reduction of hydrogen peroxide was investigated in the ranges of 1 to 15 μM with a detection limit of 0.4 μM by using chronoamperometry technique. The developed biosensor was used for the detection of hydrogen peroxide in unprocessed human plasma sample.

Dynamic Bio-Barcode Assay Enables Electrochemical Detection of a Cancer Biomarker in Undiluted Human Plasma: A Sample-In-Answer-Out Approach.

There is a need for biosensing systems that can be operated at the point-of-care (POC) for disease screening and diagnostics and health monitoring. In spite of this, simple to operate systems with the required analytical sensitivity and specificity in clinical samples, using a sample-in-answer-out approach, remain elusive. Reported here is an electrochemical bio-barcode assay (e-biobarcode assay) that integrates biorecognition with signal transduction using molecular (DNA/protein) machines and signal readout using nanostructured electrodes. The e-biobarcode assay eliminates multistep processing and uses a single step for analysis following sample collection into the reagent tube. A clinically relevant performance for the analysis of prostate specific antigen (PSA) in undiluted and unprocessed human plasma: a log-linear range of 1 ng mL-1 -200 ng mL-1 and a LOD of 0.4 ng mL-1 , was achieved. The e-biobarcode assay offers a realistic solution for biomarker analysis at the POC.

Dynamic Bio‐Barcode Assay Enables Electrochemical Detection of a Cancer Biomarker in Undiluted Human Plasma: A Sample‐In‐Answer‐Out Approach

AbstractThere is a need for biosensing systems that can be operated at the point‐of‐care (POC) for disease screening and diagnostics and health monitoring. In spite of this, simple to operate systems with the required analytical sensitivity and specificity in clinical samples, using a sample‐in‐answer‐out approach, remain elusive. Reported here is an electrochemical bio‐barcode assay (e‐biobarcode assay) that integrates biorecognition with signal transduction using molecular (DNA/protein) machines and signal readout using nanostructured electrodes. The e‐biobarcode assay eliminates multistep processing and uses a single step for analysis following sample collection into the reagent tube. A clinically relevant performance for the analysis of prostate specific antigen (PSA) in undiluted and unprocessed human plasma: a log‐linear range of 1 ng mL−1–200 ng mL−1 and a LOD of 0.4 ng mL−1, was achieved. The e‐biobarcode assay offers a realistic solution for biomarker analysis at the POC.

A novel immunosensor for the monitoring of PSA using binding of biotinylated antibody to the prostate specific antigen based on nano-ink modified flexible paper substrate: efficient method for diagnosis of cancer using biosensing technology

Prostate cancer is the most significant reason for deaths in men, outside of lung cancer. The clinical examination of cancer proteins or biomarkers is extremely significant in early examination and monitoring of recurrence of disease after treatment. Biomarkers have expanded great clinical significance owing to their extensive spectra in the identification, elimination, early diagnosis and cure of cancer. In this work, novel, ultrasensitive sandwich-type portable bio device based on citrate-capped silver nanoparticles (Citrate-AgNPs) modified graphene quantum dots (GQDs) nano ink for detection of Prostate specific antigen (PSA) was fabricated. Functionalized cysteamine with gold nanoparticles (Cys-AuNPs) was also utilized to amplify the signal. It provides a good and high external area for the immobilization biotinylated antibody of PSA in the large amount. For the first time, citrate-AgNPs-GQDs nano ink was directly written on the cellulose paper surface (ivory sheet and photographic paper) and modified by Cys-AuNPs. So, final structure of the immunodevices was completed after including of Ab1 and PSA (antigen). The immunosensors were used for the recognition of PSA by using DPVs (differential pulse voltammetry) technique. The obtained low limit of quantification (LLOQ) of the first immunodevice (ivory sheet/Citrate AgNPs-GQDs nano-ink/CysA-Au NPs/Ab1/BSA/PSA/Ab2) was 0.07 μg/L and the linear range for the calibration plot was from 0.07 to 60 μg/L. Also, the achieved LLOQ of the second immunodevice (photographic paper/Citrate AgNPs-GQDs nano-ink/Cys-Au NPs/Ab1/BSA/PSA/Ab2) was 0.05 μg/L with the linear range of 10 to 0.05 μg/L. It is noteworthy that, proposed immunoassay was effectively utilized to the monitoring of PSA glycoprotein in unprocessed human plasma sample. Obtained results show that the constructed immunosensor is able to apply as portable bio device for the clinical analysis of PSA in human plasma samples.

A novel bioassay for the monitoring of carcinoembryonic antigen in human biofluid using polymeric interface and immunosensing method.

Carcinoembryonic antigen (CEA) is a member of a family of cell surface glycoproteins. Recognition of CEA is needed to monitor the physiological status of the patient for treatment and also it is important to assess the severity of the disease. In this work, we reported a novel sandwich-type electrochemical immunosensor based on gold nanoparticles functionalized cysteamine-glutaraldehyde (AuNPs-CysA-GA) and it successfully designed to detection of the CEA biomarker in a human plasma sample. The AuNPs-CysA-GA provides a large surface area for the effective immobilization of CEA antibody, as well as it ascertains the bioactivity and stability of immobilized CEA antigens. Biotinylated-anti-CEA antibody (Ab1) was immobilized on the surface of glassy carbon electrode (GCE) modified AuNPs-CysA-GA. Also, secondary antibody (HRP-Ab2) was costed immobilized to complete the sandwich part of immunosensor. Field emission scanning electron microscope (FE-SEM and EDS), was employed to monitor the sensor fabrication procedure. The immunosensor was used for the detection of CEA using differential pulse voltammetry (DPVs) technique. The proposed interface led to enhancement of accessible surface area for immobilizing high amount of anti-CEA antibody, increasing electrical conductivity, boosting stability, and biocompatibility. Finally, the low limit of quantitation (LLOQ) of the proposed immunosensor was obtained as 7 ng/mL with the linear range of 0.001-5 μg/L. The proposed immunoassay was successfully applied for the monitoring of the CEA in unprocessed human plasma samples. Obtained results paved that the proposed bioassay can be used as a novel bioassay for the clinical diagnosis of cancer based on CEA monitoring.

An antifouling coating that enables affinity-based electrochemical biosensing in complex biological fluids.

Affinity-based electrochemical detection in complex biological fluids could enable multiplexed point-of-care diagnostics for home healthcare; however, commercialization of point-of-care devices has been limited by the rapid loss of sensitivity caused by electrode surface inactivation and biofouling. Here, we describe a simple and robust antifouling coating for electrodes consisting of a three-dimensional porous matrix of cross-linked bovine serum albumin supported by a network of conductive nanomaterials composed of either gold nanowires, gold nanoparticles or carbon nanotubes. These nanocomposites prevent non-specific interactions while enhancing electron transfer to the electrode surface, preserving 88% of the original signal after 1 month of exposure to unprocessed human plasma, and functionalization with specific antibodies enables quantification of anti-interleukin 6 in plasma with high sensitivity. The easy preparation, stability and simplicity of this nanocomposite allow the generation of electrochemical biosensors that can operate in complex biological fluids such as blood plasma or serum.

Simultaneous Detection of Morphine and Codeine in the Presence of Ascorbic Acid and Uric Acid and in Human Plasma at Nafion Single-Walled Carbon Nanotube Thin-Film Electrode.

In clinical settings, the dosing and differential diagnosis of the poisoning of morphine (MO) and codeine (CO) is challenging due to interindividual variations in metabolism. However, direct electrochemical detection of these analytes from biological matrices is inherently challenging due to interference from large concentrations of anions, such as ascorbic acid (AA) and uric acid (UA), as well as fouling of the electrode by proteins. In this work, a disposable Nafion-coated single-walled carbon nanotube network (SWCNT) electrode was developed. We show facile electron transfer and efficient charge separation between the interfering anions and positively charged MO and CO, as well as significantly reduced matrix effect in human plasma. The Nafion coating alters the voltammetric response of MO and CO, enabling simultaneous detection. With this SWCNT/Nafion electrode, two linear ranges of 0.05–1 and 1–10 μM were found for MO and one linear range of 0.1–50 μM for CO. Moreover, the selective and simultaneous detection of MO and CO was achieved in large excess of AA and UA, as well as, for the first time, in unprocessed human plasma. The favorable properties of this electrode enabled measurements in plasma with only mild dilution and without the precipitation of proteins.

Immunosensing of breast cancer tumor protein CA 15-3 (carbohydrate antigen 15.3) using a novel nano-bioink: A new platform for screening of proteins in human biofluids by pen-on-paper technology.

Early stage diagnosis of breast cancer by monitoring the proteins in human biological fluids is the best method and the first section toward efficient therapy, delaying metastasis and hindrance mortality. In this study, graphite ink was synthesized and combined with CA 15-3 antibody in order to develop a novel immunosensor for detection of CA 15-3, breast cancer biomarker. Conductivity of bioink was examined by designing various conductive patterns on paper using a rollerball pen. Electrochemical behavior of engineered immunosensor was evaluated through employing sensitive diagnostic technique, DPV (differential pulse voltammetry). Under optimal conditions, the linear concentration range and low limit of quantification (LLOQ) of designed immunosensor was 15-250 U/mL and 15 U/mL, respectively. The process was successfully applied to assay of breast cancer biomarker (CA15-3) in unprocessed human plasma specimens.

A novel paper based immunoassay of breast cancer specific carbohydrate (CA 15.3) using silver nanoparticles-reduced graphene oxide nano-ink technology: A new platform to construction of microfluidic paper-based analytical devices (μPADs) towards biomedical analysis

The technology of ink-jet printing has been an appealing alternative to traditional photolithography for fabricating flexible and elastic circuits, having low-cost and ease of control. In this work, a novel, unique and sensitive immunoassay designed based on application of silver nanoparticles-reduced graphene oxide nanocomposite (Ag/RGO) ink for fabrication of paper based immunosensor, Cysteamine coped gold nanoparticles (CysA/Au NPs) as a signal amplification element for determination of CA15-3 protein. Fully electrochemically methodology was applied to prepare a new transducer based on the Ag/RGO surface which provides high surface area for CA15-3 antibody immobilization in high amount. Under optimized experimental conditions, the Ag-RGO/CysA-Au NPs based immunosensor shows good specificity and sensitivity. The obtained LLOQ of immunosensor for CA 15-3 biomarker was 15 U/mL as well as calibration curve was linear in the range of 15–125 U/mL. The technique was successfully employed assay of CA15-3 biomarker in unprocessed human plasma samples.

Ultrasensitive bioassay of epitope of Mucin-16 protein (CA 125) in human plasma samples using a novel immunoassay based on silver conductive nano-ink: A new platform in early stage diagnosis of ovarian cancer and efficient management

Ovarian cancer (OC) is known to be one of the most lethal malignancies associated with women disease. The CA-125 protein is a repetitive epitope of MUC-16, which plays key role in enhancing the proliferation of cancer cells and inhibiting anticancer immune responses. It is the most widely used biomarker for early stage diagnosis of OC. Also it is the only serum marker which currently used in clinical diagnosis. Monitoring of CA-125 protein in the serum sample is also valuable in evaluating the response of ovarian cancer to treatment. In this research, a novel immunoassay based on immobilization of CA-125 antibody on the biointerface of silver nanoparticles modified graphene quantum dots ink (Ag NPs-GQDs) was successfully designed to recognition of CA-125 protein in a human plasma sample. The supplied immunoassay presents the proper ability to detect and determine the amount of CA-125 biomarker in low concentrations of CA-125 biomarker. The proposed immunosensor was employed for the detection of CA-125 using differential pulse voltammetry (DPVs) and square wave voltammetry (SWVs) techniques. The proposed interface leads to enhancement of accessible surface area for immobilizing a high amount of anti-CA-125 antibody, increasing electrical conductivity, boosting stability, catalytic properties and biocompatibility. Under the optimized operating conditions, the low limit of quantitation (LLOQ) for the proposed immunosensor was recorded as 0.01 U/mL, which this evaluation was performed at highly linear range of 0.01–400 U/mL. The proposed immunoassay was successfully applied for the monitoring of CA-125 in unprocessed human plasma samples.

Sensitive detection and determination of benzodiazepines using silver nanoparticles-N-GQDs ink modified electrode: A new platform for modern pharmaceutical analysis

In this work, a unique conductive nano-ink based on silver nanoparticle‑nitrogen doped graphene quantum dots (Ag/N-GQD) was synthesized and utilized for development of a novel, unique and sensitive biosensor for determination of alprazolam, chlordiazepoxide bis, diazepam, oxazepam and clonazepam. Electrochemical deposition, as a well-controlled synthesis procedure, has been utilized for subsequently layer-by-layer preparation of Ag/N-GQD nano-ink on a chitosan (CS) electropolymerized on the surface of gold electrode using cyclic voltammetry techniques in the regime of −1 to +1 V. The modified electrode appeared as an efficient electroactive interface for detection of selected benzodiazepines by using cyclic voltammetry, differential pulse voltammetry, and square wave voltammetry. The prepared sensor showed a noticeably good electro-activity for electrooxidation of alprazolam, chlordiazepoxide bis, diazepam, oxazepam and clonazepam than bare CS and Ag/N-GQD modified electrodes. Enhancement of peak currents is ascribed to the fast heterogeneous electron transfer kinetics that arise from the synergistic coupling between unique features of CS such as high permeability, excellent film-forming capability, good adhesion, cheapness, nontoxicity, and a susceptibility to chemical modifications, Ag/N-GQD nano-ink as high electrical conductivity. Finally, application of the new sensor for determination of selected benzodiazepine group drugs in unprocessed human plasma samples was studied. In general, the simultaneous attachment of Ag/N-GQD nano-ink to structure of CS provides new opportunities toward biomedical analysis and the personal healthcare.

Application of bioactive cyclic oligosaccharide on the detection of doxorubicin hydrochloride in unprocessed human plasma sample: A new platform towards efficient chemotherapy

In this study, a novel nano-composite based on poly‑taurine/beta‑cyclodextrin and graphene quantum dot used for the modification of gold electrode by simple electrodeposition approach. The prepared interface was applied for the sensing of doxorubicin hydrochloride (DOX) in human plasma samples without any pretreatment procedure. The synergistic influence of poly-taurine, beta-cyclodextrin, and graphene quantum dot not only remarkably enhances electroactivity, but also speeds up the electron shift between DOX and the electrode substrates. Cyclic voltammetry results indicated that the DOX is oxidized via two electrons and two protons process at the optimum pH = 4 using poly-taurine/beta-cyclodextrin and graphene quantum dots thin film modified gold electrode. More momentously electrostatic repulsion takes place between the prepared polymeric film and DOX resulting in the signal amplification on oxidation of this drug and lowering its overpotential and thereby sensitive and selective detection of DOX in real samples. The apparent electron transfer rate constant and transfer coefficients were determined by cyclic voltammetry. Using differential-pulse voltammetric (DPV) technique sensitive determination of DOX in human plasma samples was performed with wide dynamic range and good limit of detection.

Ultrasensitive immunoassay of glycoprotein 125 (CA 125) in untreated human plasma samples using poly (CTAB‑chitosan) doped with silver nanoparticles

CA 125 is a Mucin glycoprotein and its concentration in human serum correlates with a woman's risk of developing ovarian cancer and also indicates response to therapy in diagnosed patients. Accurate detection of this large, complex protein in patient biofluids is of great clinical relevance. In this work, an innovative immunoassay for quantitation of CA 125 based on signal amplification strategy was proposed. In this work, Horseradish peroxidase (HRP) labeled CA 125-antibody (anti-CA 125) was immobilized onto a green and biocompatible nanocomposite containing poly cetyl trimethyl ammonium bromide (P (CTAB) as conductive matrix, chitosan (CS) as biocompatible agent and sliver nanoparticles (Ag NPs) as signal amplification element. Therefore, a novel nanocomposite film based P (CTAB-CS) and Ag NPs was exploited to develop a highly sensitive immunosensor for detection of CA 125 protein. Importantly, Ag NPs prepared by electrodeposition method which lead to compact morphology. Fully electrochemical methodology was used to prepare a new transducer on a glassy carbon surface which provided a high surface area to immobilize a high amount of the anti-CA 125. The surface morphology of electrode was characterized by high-resolution field emission scanning electron microscope (FE-SEM) and energy dispersive spectroscopy (EDS). The immunosensor was employed for the detection of CA 125 using cyclic voltammetry (CV) and differential pulse voltammetry (DPVs) techniques. Under optimized condition the calibration curve for CA 125 concentration by SWV and DPV was linear in 0.01–400 U/mL with lower limit of quantification of 0.001 U/mL. The method was successfully applied assay of the CA 125 in unprocessed human plasma samples.